In order for a piece of circular, dsDNA to be propagated in bacteria, it needs to be replicated by host machinery. There is a sequence in the plasmid that directs the cell to begin replication. Important considerations are host range, compatibly, and copy number. The host range refers to what species of bacteria will recognize the origin of replication and thus allow for replication. The compatibility refers to a plasmid's ability to coexist with another plasmid in the same cell. Copy number refers to the average or expected number of copies of the plasmid per cell.

In order for a piece of circular, dsDNA to be propagated in bacteria, it needs to be replicated by host machinery. There is a sequence in the plasmid that directs the cell to begin replication. Important considerations are host range, compatibly, and copy number. The host range refers to what species of bacteria will recognize the origin of replication and thus allow for replication. The compatibility refers to a plasmid's ability to coexist with another plasmid in the same cell. Copy number refers to the average or expected number of copies of the plasmid per cell.

There are three main mechanisms for plasmid replication: Rolling Circle, Strand Displacement, and Theta.

There are three main mechanisms for plasmid replication: Rolling Circle, Strand Displacement, and Theta.

-

*Strand Displacement...

+

====Strand displacement replication====

+

RepC binds repeat sequences recruits RepA ( a helicase) to melt an AT rich region. This exposes two single stranded origins ''ssiA'' and ''ssiB.'' RepB polymerizes primers for these origins. DNA polymerization follows in each direction, meanwhile displacing the non-template stand.

-

*In Rolling Circle Replication, a nick is made in the minus strand at the "plus strand origin" of a dsDNA plasmdid. The free 3'OH is extended, displacing as it progresses. The displaced minus strand then serves as a template for replication from "minus strand origins." If minus stand origins are defective, then ssDNA can accumulate. This mechanism is found in ''Staphylococcus aureus'' and ''Streptomyces lividans'' as well as many bacteriophages. (Genes and Genetic Elements)

+

Strand displacement is associated with broad host range vectors, possibly because it does not require any of the normal host machinery (DnaA, DnaB, DnaC, and DnaG)

-

*Theta replication is the most common form of DNA replication, including most plasmids as well as chromosomes. RNA serves a primer, DNA is polymerized in one or both directions. In the first case, a single fork circumnavigates the entire plasmid until the origin is reached, and daughter plasmids separate. In bidirectional replication, two forks propagate and meet on the far side of the plasmid before resolution.

+

====Rolling circle replication====

+

A nick is made by the Rep protein at the "double strand origin" of a dsDNA plasmdid. The free 3'OH is extended, displacing as it progresses. After one unit length of displacement, replication is terminated, yielding one dsDNA plasmid and ssDNA of one unit length. The displaced strand then serves as a template for replication from a "single strand origins." Since each strand is replicated independently, it is possible for the ssDNA form to accumulate.

-

===Host Range===

+

This mechanism is found in gram-positive bacteria like ''Staphylococcus aureus'' and ''Streptomyces lividans'' as well as many bacteriophages.

-

Plasmids are classified as having a narrow or broad host range. For example, ColE1 is limited to ''E. coli'' and a few close relatives, while RK2 plasmids can be used in most gram-negative bacteria. Plasmids from gram-positive bacteria tend to function well in other gram-positive bacteria.

+

-

===Compatibility Groups===

+

====Theta replication====

-

If two plasmids have the same (or very similar) origins of replication, they will compete with each other for replication machinery. This results in an unstable situation. If the two plasmids posses different selectable markers, this can be maintained for several generations, but eventually one of the plasmids will be lost. For scenarios in which multiple plasmids are necesary, one must be careful to choose plasmids will compatible origins. The most common dual-plasmid pair is ColE1/p15A. The most common plasmid triplet is ColE1/p15A/pSC101.

+

DnaA (often with the help of other proteins) binds the origin at DnaA boxes. This promotes melting of the orgin. This allows DnaC to load to DnaB helicase, opening the origin further. DnaG is then recuited to form a short RNA primer.

+

DNA polymerase III extends this primter. If there is only one leading primer, a single fork circumnavigates the entire plasmid until the origin is reached, and daughter plasmids separate. In bidirectional replication, two forks propagate and meet on the far side of the plasmid before resolution.

-

*ColE1: pUC, pMB1, pBR322, pGEM, pET, pUC, pQE, pMAL, pGEX

+

Theta is the most common form of DNA replication, including most plasmids as well as chromosomes. It is particularly associated with gram-negative bacteria. ColE1, P15A, RK2, F, and P1 all use theta replication.

-

*p15A: pBad, pACYC

+

-

*pSC101

+

==Host range==

+

Plasmids are classified as having a narrow or broad host range.

+

*ColE1 and pMB1 are limited to ''E. coli'' and a few close relatives,

+

*RK2 plasmids can be used in most gram-negative bacteria.

+

*RSF1010 can use used in most gram-negative bacteria, and some gram-positive

+

*Plasmids from gram-positive bacteria tend to function well in other gram-positive bacteria.

+

+

==Compatibility groups==

+

If two plasmids have the same (or very similar) origins of replication, they will compete with each other for replication machinery. This results in an unstable situation. If the two plasmids posses different selectable markers, this can be maintained for several generations, but eventually one of the plasmids will be lost. For scenarios in which multiple plasmids are necesary, one must be careful to choose plasmids will compatible origins. The most common dual-plasmid pair is ColE1(or pMB1) and p15A. The most common plasmid triplet is ColE1 (or pMB1),p15A, and pSC101. Tolia and Joshua-Tor suggest the following groups:

+

+

*ColE1/pMB1 (eg pET, pUC, pBR322, pGEX, pMAL)

+

*P15A (eg pBad, pACYC)

*CloDF13

*CloDF13

-

*ColA

+

*ColA

*RSF1030

*RSF1030

-

===Copy Number===

+

==Copy number==

-

*ColE1: 20-700 copies

+

An important consideration in choosing what plasmid backbone to use is the copy number. For example, cloning is best done with a high copy plasmid (e.g. pUC) as plasmid preps will have a higher yield. Expressing a toxic gene is better from a low to medium copy plasmid(e.g. pET which uses the pBR322 origin), as there are fewer copies.

-

**pUC: 500-700 copies

+

+

*ColE1: 15-20 copies

+

*pMB1: 20-700 copies

+

**pUC: 500-700 copies

**pBR322: ~20 copies

**pBR322: ~20 copies

*pSC101: ~5 copies

*pSC101: ~5 copies

-

*p15A: 10-12 copies

+

*P15A: 10-12 copies

-

*R1:

+

*RK2: 4-7 copies

-

*RK2:4-7

+

-

*R6K:

+

*F1: ~1 copy

*F1: ~1 copy

-

*CloDF13: 20-40

+

*CloDF13: 20-40 copies

-

*ColA: 20-40

+

*ColA: 20-40 copies

-

*RSF1030: >100

+

*RSF1030: >100 copies

-

P1:1

+

*P1: ~1 copy

+

*R6K: 15-30 copies

+

+

===Control of initiation/copy number===

+

There are several mechanisms by which copy number is controlled. In all cases, some negative-regulating element (RNA or protein) is expressed from the plasmid. As the plasmid concentration increases, so too does the negative regulator. This provides a negative feedback, which stabilizes the copy number. Two plasmids that are regulated by each other's regulator will not be compatible.

+

+

====RNA regulation====

+

ColE1/pMB1: The origin contains regions promoting the synthesis of RNA I and RNA II. RNA II hybrizes to the DNA, yielded a DNA/RNA hybrid which can serve as a substrate for RNaseH. Digestion of RNA II by RNaseH yields the primer for replication. RNA I binds and sequesters RNA II, so it is unavailable for RNAse H digestion. As the copy number increases, so does the concentration of RNA I. This provides a negative feedback for replication, and sets the average number of plasmids per cell.

+

+

Additionally, The Rop protein helps lower the copy number, by stabilizing the RNA I/ RNA II duplex. Deletion of Rop, as well as a point mutation that weakens the RNA I and RNA II duplex, accounts for the higher copy of pUC (a pMB1 derivatives)

+

+

P15A, ColA, RSF1030, and CloDF13 are similar, but with versions of RNA I and RNA II that sufficiently different to allow for compatibility.

+

+

====RNA and protein regulation====

+

On the R1 plasmid, OriR is bound by RepA, thus promoting replication by recruiting DnaA. RepA can be expressed from two different promoter. A proximal promoter (pRepA) drives only RepA while a distal promoter (pCopB) drives both CopB and RepA. CopB represses pRepA, thus once there are enough plasmids around, CopB levels become high enough to limit RepA expression to pCopB promoter. plasmid encoded CopA is completmentary, and thus binds to the 5' end of the transcript originating from the pCopB promoter. The dsRNA is a substrate for the processive RNase III.

+

+

====Iteron regulation====

+

Like the above examples, pSC101's replication is positively regulated by RepA binding the origin. RepA is also used to control copy number, by two mechanisms.

+

+

Firstly, RepA negatively regulates its own transcription, thus the RepA protein levels (and its ability to promote replication) is confined to narrow limits.

+

+

Secondly, The plasmid contains several (3-7) repeats of a 17-22bp sequence called iteron sequences. RepA binds the iterons, and at higher plasmid conncentration, this can lead to "handcuffing" of two plasmids. Interestingly, adding extra iteron sequences on other plasmids can reduce the copy number by this handcuffing mechanism.

+

+

F, RK6, P1, RK2, and RP4 also use iterons, but the regulating protein and origins differ.

-

===Control of Initiation/copy number===

+

pETcoco is an interesting plasmid, made by Novagen. It can be maintained as a single copy plasmid using the origin and positive regulator from the F plasmid (oriS and RepE). It can be swiched to a medium copy plasmid using the machinery from the RK2 plasmid (oviV and trfA). The switch is achieved by the induction of the trfA protein, which binds and iteron on oriV, thus promoting initiation from this origin by aiding in melting and recruitment of DnaB.

-

*ColE1: Does not require plasmid encoded initiator protein, does require DnaA binding. 1kb. Includes (1) region promoting the synthesis of RNA II, which is the primer for the leading strand (2) sequences that promote stable hybridization of RNA II to DNA (3) sequences that promote RNAse digestion of RNA II, yielding primer for leading strand. RNA I is also transcribed, and sequesters RNA II, so it is unavailable for RNAse H.

+

==Other extra-chromosomal bodies==

-

*p15A: similar to ColE1 but different RNAs

+

===BACs===

-

*pSC101: iterons

+

BACs (bacterial artificial chromosomes) are based on the single-copy F origin. It is capable of maintaining inserts greater than 300kb. Key to its stability are the par elements. ParA and parB are plasmid encoded proteins that help ensure that the each daughter cell gets one copy of the BAC during binary fission.

-

*R1: oriR is bound by RepA promoting initiation. No iterons. Copy number controlled by copA and copB. copA is in RNA that binds the 5’ end of RepA RNA and prevents translation. CopB blocks repA transcription.

About 10% of the sequenced bacteria contain large "second chromosome," dubbed chromids. By definition, chromids are the second largest replicon in a bacteria, has a plasmid type maintenance and replication system, have a GC content similar to chromosome, and carry core genes that one are found on the chromosome in other species. Chromids vary in size from 300kb to 3Mb.

Possible explanations for chromids are that 1) they contain essential genes are "frozen accidents" 2) spliting the essential genes onto two bodies allows for faster replication 3) having some genes on a second body allows for differential regulation.

==References==

==References==

<biblio>

<biblio>

-

#Solar1998 pmid=PMC98921

+

#Solar1998 pmid=9618448

-

</biblio>

+

//Review

+

#Lin-Chao1992 pmid=1283002

+

//Regarding the copy number of pUC vs pBR322

+

#(Genes and Genetic Elements)

+

#Tolia2006 pmid=16369554

+

//regarding plasmid compatibility.

+

#Seizer1983 pmid=6186390

+

//regarding the differences between RNA I and RNA II sequences in P15A, ColE1, RSF1030, CloDF13, and pBR322.

Plasmid Replication

In order for a piece of circular, dsDNA to be propagated in bacteria, it needs to be replicated by host machinery. There is a sequence in the plasmid that directs the cell to begin replication. Important considerations are host range, compatibly, and copy number. The host range refers to what species of bacteria will recognize the origin of replication and thus allow for replication. The compatibility refers to a plasmid's ability to coexist with another plasmid in the same cell. Copy number refers to the average or expected number of copies of the plasmid per cell.

There are three main mechanisms for plasmid replication: Rolling Circle, Strand Displacement, and Theta.

Strand displacement replication

RepC binds repeat sequences recruits RepA ( a helicase) to melt an AT rich region. This exposes two single stranded origins ssiA and ssiB. RepB polymerizes primers for these origins. DNA polymerization follows in each direction, meanwhile displacing the non-template stand.

Strand displacement is associated with broad host range vectors, possibly because it does not require any of the normal host machinery (DnaA, DnaB, DnaC, and DnaG)

Rolling circle replication

A nick is made by the Rep protein at the "double strand origin" of a dsDNA plasmdid. The free 3'OH is extended, displacing as it progresses. After one unit length of displacement, replication is terminated, yielding one dsDNA plasmid and ssDNA of one unit length. The displaced strand then serves as a template for replication from a "single strand origins." Since each strand is replicated independently, it is possible for the ssDNA form to accumulate.

This mechanism is found in gram-positive bacteria like Staphylococcus aureus and Streptomyces lividans as well as many bacteriophages.

Theta replication

DnaA (often with the help of other proteins) binds the origin at DnaA boxes. This promotes melting of the orgin. This allows DnaC to load to DnaB helicase, opening the origin further. DnaG is then recuited to form a short RNA primer.

DNA polymerase III extends this primter. If there is only one leading primer, a single fork circumnavigates the entire plasmid until the origin is reached, and daughter plasmids separate. In bidirectional replication, two forks propagate and meet on the far side of the plasmid before resolution.

Theta is the most common form of DNA replication, including most plasmids as well as chromosomes. It is particularly associated with gram-negative bacteria. ColE1, P15A, RK2, F, and P1 all use theta replication.

Host range

Plasmids are classified as having a narrow or broad host range.

ColE1 and pMB1 are limited to E. coli and a few close relatives,

RK2 plasmids can be used in most gram-negative bacteria.

RSF1010 can use used in most gram-negative bacteria, and some gram-positive

Plasmids from gram-positive bacteria tend to function well in other gram-positive bacteria.

Compatibility groups

If two plasmids have the same (or very similar) origins of replication, they will compete with each other for replication machinery. This results in an unstable situation. If the two plasmids posses different selectable markers, this can be maintained for several generations, but eventually one of the plasmids will be lost. For scenarios in which multiple plasmids are necesary, one must be careful to choose plasmids will compatible origins. The most common dual-plasmid pair is ColE1(or pMB1) and p15A. The most common plasmid triplet is ColE1 (or pMB1),p15A, and pSC101. Tolia and Joshua-Tor suggest the following groups:

ColE1/pMB1 (eg pET, pUC, pBR322, pGEX, pMAL)

P15A (eg pBad, pACYC)

CloDF13

ColA

RSF1030

Copy number

An important consideration in choosing what plasmid backbone to use is the copy number. For example, cloning is best done with a high copy plasmid (e.g. pUC) as plasmid preps will have a higher yield. Expressing a toxic gene is better from a low to medium copy plasmid(e.g. pET which uses the pBR322 origin), as there are fewer copies.

ColE1: 15-20 copies

pMB1: 20-700 copies

pUC: 500-700 copies

pBR322: ~20 copies

pSC101: ~5 copies

P15A: 10-12 copies

RK2: 4-7 copies

F1: ~1 copy

CloDF13: 20-40 copies

ColA: 20-40 copies

RSF1030: >100 copies

P1: ~1 copy

R6K: 15-30 copies

Control of initiation/copy number

There are several mechanisms by which copy number is controlled. In all cases, some negative-regulating element (RNA or protein) is expressed from the plasmid. As the plasmid concentration increases, so too does the negative regulator. This provides a negative feedback, which stabilizes the copy number. Two plasmids that are regulated by each other's regulator will not be compatible.

RNA regulation

ColE1/pMB1: The origin contains regions promoting the synthesis of RNA I and RNA II. RNA II hybrizes to the DNA, yielded a DNA/RNA hybrid which can serve as a substrate for RNaseH. Digestion of RNA II by RNaseH yields the primer for replication. RNA I binds and sequesters RNA II, so it is unavailable for RNAse H digestion. As the copy number increases, so does the concentration of RNA I. This provides a negative feedback for replication, and sets the average number of plasmids per cell.

Additionally, The Rop protein helps lower the copy number, by stabilizing the RNA I/ RNA II duplex. Deletion of Rop, as well as a point mutation that weakens the RNA I and RNA II duplex, accounts for the higher copy of pUC (a pMB1 derivatives)

P15A, ColA, RSF1030, and CloDF13 are similar, but with versions of RNA I and RNA II that sufficiently different to allow for compatibility.

RNA and protein regulation

On the R1 plasmid, OriR is bound by RepA, thus promoting replication by recruiting DnaA. RepA can be expressed from two different promoter. A proximal promoter (pRepA) drives only RepA while a distal promoter (pCopB) drives both CopB and RepA. CopB represses pRepA, thus once there are enough plasmids around, CopB levels become high enough to limit RepA expression to pCopB promoter. plasmid encoded CopA is completmentary, and thus binds to the 5' end of the transcript originating from the pCopB promoter. The dsRNA is a substrate for the processive RNase III.

Iteron regulation

Like the above examples, pSC101's replication is positively regulated by RepA binding the origin. RepA is also used to control copy number, by two mechanisms.

Firstly, RepA negatively regulates its own transcription, thus the RepA protein levels (and its ability to promote replication) is confined to narrow limits.

Secondly, The plasmid contains several (3-7) repeats of a 17-22bp sequence called iteron sequences. RepA binds the iterons, and at higher plasmid conncentration, this can lead to "handcuffing" of two plasmids. Interestingly, adding extra iteron sequences on other plasmids can reduce the copy number by this handcuffing mechanism.

F, RK6, P1, RK2, and RP4 also use iterons, but the regulating protein and origins differ.

pETcoco is an interesting plasmid, made by Novagen. It can be maintained as a single copy plasmid using the origin and positive regulator from the F plasmid (oriS and RepE). It can be swiched to a medium copy plasmid using the machinery from the RK2 plasmid (oviV and trfA). The switch is achieved by the induction of the trfA protein, which binds and iteron on oriV, thus promoting initiation from this origin by aiding in melting and recruitment of DnaB.

Other extra-chromosomal bodies

BACs

BACs (bacterial artificial chromosomes) are based on the single-copy F origin. It is capable of maintaining inserts greater than 300kb. Key to its stability are the par elements. ParA and parB are plasmid encoded proteins that help ensure that the each daughter cell gets one copy of the BAC during binary fission.

Chromids

About 10% of the sequenced bacteria contain large "second chromosome," dubbed chromids. By definition, chromids are the second largest replicon in a bacteria, has a plasmid type maintenance and replication system, have a GC content similar to chromosome, and carry core genes that one are found on the chromosome in other species. Chromids vary in size from 300kb to 3Mb.

Possible explanations for chromids are that 1) they contain essential genes are "frozen accidents" 2) spliting the essential genes onto two bodies allows for faster replication 3) having some genes on a second body allows for differential regulation.